Recombinant DNA technology has become a tool for studying the relationship between protein structure and function. In particular, properties of a given protein can be changed by engineering mutations in the gene encoding the protein. Recently, there has been renewed interest in the practice of screening and selecting among random mutants for structurally altered proteins with properties of interest (Wells et al. (1992) Curr. Opin. Struct. Biol. 2:597-604; Gram et al. (1992) Proc. Natl. Acad. Sci. USA 89:3576-3580; Delagrave et al. (1993) Bio/Technology 11:1548-1552).
It has become possible to construct large pools of random mutants which encompass virtually every possible single mutant of a given protein using modern techniques of molecular biology. For example, there are 9,500 different single mutants of a 500 amino acid protein; using recombinant DNA methods, one can generate a population of 75,000 independent clones which includes every possible single mutant (Cormack et al. (1993) Science 262:244-248). However, due to limits in transformation technologies, which typically yield 10.sup.7 transformants per experiment, it is only possible to sample a very small fraction of all possible double mutants without resorting to intermediate selection steps. (A protein of 500 amino acids has on the order of 10.sup.8 possible double mutants.)
A potential exception to this is exemplified by the work of Winter and coworkers (Waterhouse et al. (1993) Nucleic Acids Research 21:2265-2266), who utilized an in vivo site-specific recombination system to combine light chain antibody genes with heavy chain antibody genes for expression in a phage display system. Theoretically, this strategy could be used to generate a library of as many as 10.sup.14 (10.sup.7 .times.10.sup.7) F.sub.AB (or phab) expressing clones.
The general technique for identifying protein variants with desired characteristics, as summarized in Delagrave et al., supra, involves exhaustively sampling all possible single mutants of a target protein for any property of interest. (This strategy can be modified to incorporate a small subset of double mutants.) Candidate mutations that emerge from such screens are then combinatorially assembled into ensembles of multiple mutants, which can be iteratively screened for further improvement in the property of interest.
In many applications, however, a sufficiently sensitive method to screen more than 10.sup.7 mutants in a single step is unavailable. For example, in situations where screening involves affinity "panning", the isolation of a desirable mutant is limited by the resolution of a chromatography column. It is by no means clear that the mere availability of a mutant library containing more than 10.sup.7 clones would result in the isolation of a superior mutant in a single step. In such situations an iterative mutagenesis and screening strategy, which gradually converges to an acceptable solution in sequence space, may be unavoidable.
Disclosure
In order to address the above-described deficiencies in the art, the inventors herein disclose a novel method (called "recombination-enhanced mutagenesis") for in vivo mutant construction that can exceed the limitation of 10.sup.7 mutant proteins by several orders of magnitude, thereby facilitating a more exhaustive search of protein sequence space for useful double and possibly even triple mutants. Since this method does not require the introduction of a specific recombination site, it can be used to randomly recombine multiple mutations whose relative positions in the wild-type encoding nucleotide sequence are unknown.
Using recombination-enhanced mutagenesis it is possible to generate arbitrarily large libraries of multiple mutants in a protein of interest. Therefore, given a sufficiently "clean" screening/selection technique, such as in vivo selection of a metabolically essential protein, it is be possible to sample virtually all possible double (and even triple) mutants in a single selection step.
Recombination-enhanced mutagenesis also eliminates the need for iterative mutagenesis and cloning. This is possible because, after a first round of "panning," the enriched mutant pool can be recombined with the initial mutant pool to generate a second generation mutant pool. In other words, only one high quality mutant pool needs to be constructed via in vitro mutagenesis.
Accordingly, in one embodiment, the invention is directed to a method of generating multiple protein variants. The method comprises:
a) providing first and second sets of allelic mutants, wherein said first set of allelic mutants comprises at least one recipient pool of mutant encoding nucleotide sequences and said second set of allelic mutants comprises at least one donor pool of mutant encoding nucleotide sequences; PA1 b) cloning each recipient pool of mutant encoding nucleotide sequences into a recipient vector; PA1 c) transforming or transducing the recipient vector into a host cell to generate a pool of recipient cells; PA1 d) cloning each donor pool of mutant encoding nucleotide sequences into a donor vector selected from the group consisting of phagemids and cosmids; PA1 e) transforming or transducing the donor vector into a host cell to generate a pool of donor cells; PA1 f) infecting the pool of donor cells with a helper phage to generate a mixture of donor phage particles, wherein said mixture comprises donor phage particles which contain said helper phage genome and phage particles which contain donor vectors having a mutant encoding nucleotide sequence; PA1 g) clonally amplifying the pools of donor phage and recipient cells; PA1 h) transducing the recipient cells with the mixture of donor phage particles to generate a set of recombinants; and PA1 i) screening the recombinants.
In a second embodiment, the invention is directed to a population of multiple protein variants prepared by the method disclosed herein.
These and other embodiments of the subject invention will readily occur to those of ordinary skill in the art in view of the disclosure herein.